Endocrinology, doi:10.1210/en.2006-1442
Endocrinology Vol. 148, No. 3 1167-1170
Copyright © 2007 by The Endocrine Society
A Topical Parathyroid Hormone/Parathyroid Hormone-Related Peptide Receptor Antagonist Stimulates Hair Growth in Mice
Joshua D. Safer,
Swapna Ray and
Michael F. Holick
Vitamin D, Skin, and Bone Research Laboratory, Section of Endocrinology, Diabetes, and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
Address all correspondence and requests for reprints to: Joshua D. Safer, M.D., Section of Endocrinology, Diabetes, and Nutrition, Boston University School of Medicine, 715 Albany Street, Room M-1016, Boston, Massachusetts 02118. E-mail: jsafer{at}bu.edu.
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Abstract
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One of the biggest hurdles in developing peptides for the treatment of hair growth disorders is that there has been no effective method of delivering them topically. Murine hair growth can be stimulated with ip injections of the PTH/PTHrP receptor antagonist, PTH (7–34). We sought to determine whether we could deliver PTH (7–34) topically and achieve stimulation of hair growth. We prepared a topical cream by mixing PTH (7–34) into a liposome vehicle (Novasome A). We applied the cream daily to the backs of 5-wk-old female SKH-1 hairless mice for 1 wk. After the 1 wk of treatment, there was marked stimulation of hair growth in the SKH-1 hairless mice. Relative to controls, mice treated with PTH (7–34) had 216% longer hairs (P < 0.001), 40% more visible hairs (P < 0.001), and 43% more hair follicles stained with 5-bromo-2'-deoxyuridine (P < 0.01). A unique aspect of skin is the possibility to directly target it via topical treatment. Our study is the first to report the hair-stimulating effect of a PTH/PTHrP receptor antagonist topically applied to skin in vivo. Thus, we introduce a novel paradigm to develop topical PTH analogs for treating disorders of hair growth.
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Introduction
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IN ADDITION TO being a product of tumors that are associated with humoral hypercalcemia of malignancy (1), PTHrP is also expressed by a variety of normal and neoplastic tissues, including the skin and hair follicles (2, 3).
Although the physiological role of PTHrP in the skin is not well understood, mounting evidence suggests that this peptide plays an important role in epidermal proliferation and differentiation (4, 5, 6). The PTH/PTHrP receptor agonist, PTH 1–34, inhibited epidermal proliferation in cultured keratinocytes and SKH-1 hairless mice (7). The PTH/PTHrP receptor antagonist, PTH (7–34), reversed the agonist’s antiproliferative effect in vitro. In mice, ip PTH (7–34) stimulated 3H-thymidine incorporation into epidermis in a dose-dependent manner, increased visible hair number, increased hair length, and advanced telogen hair follicles into anagen (6, 8).
The current project was undertaken to determine whether we could deliver PTH (7–34) topically and achieve hair growth stimulation in that fashion.
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Materials and Methods
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Preparation and application of topical PTH (7–34) cream
We prepared a topical cream by mixing human PTH (7–34) (Bachem, Torrance, CA) into a liposome vehicle (Novasome A; IGI Inc., Buena, NJ) as previously described (9, 10). Novasome A is a commercial liposome vehicle that contains ethoxydiglycol (25%), glyceryl dilaurate (8%), propylene glycol dicaylate (4%), cholesterol (1.5%), and cetearyl alcohol/cetearyl glucoside (1%). The PTH (7–34) cream (10 µg in 30 µl vehicle) was applied daily to a defined 3 x 3 cm region in the midline backs of five 5-wk-old female SKH-1 hairless mice (Charles River, Boston, MA). Five age-, size-, and sex-matched control mice were treated with vehicle alone.
All mouse experimentation was approved by the Animal Use and Care Committee at Boston University School of Medicine.
Measurement of mouse hair characteristics
After 1 wk of treatment, all mice were killed with 10 µm full-thickness dorsal skin samples taken, fixed in formalin, embedded in paraffin, and longitudinally sectioned. Random 2-mm segments of epidermis were delineated on the histology sections, epidermal thickness measured, and the number of hairs protruding counted. Photographs of the mice were taken before the animals were killed and 10 random hairs visible in cross-section measured as previously described (10).
Evaluation of mouse epidermal proliferation
Epidermal proliferation was assessed by measuring 3H-thymidine incorporation and 5-bromo-2'-deoxyuridine (BrdU; Roche Molecular Biochemicals, Mannheim, Germany) incorporation into DNA. Twenty-four hours before the animals were killed, each mouse received ip 3H-thymidine as previously described (10). The mice received additional 3H-thymidine along with BrdU 3 h before the animals were killed. 3H-thymidine incorporation into DNA was assessed with a ß-counter and corrected for DNA content. Skin samples described above were stained for BrdU as previously described (10). BrdU data are reported as the number of cell nuclei stained per millimeter epidermis or the percentage of hair follicles staining positive for BrdU as indicated.
Statistical analysis
For the wound-healing study, statistical analysis was performed with Student’s t test. Data are presented with SEM.
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Results
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Topical PTH (7–34) stimulated hair growth
Daily topical application of 10 µg PTH (7–34) for 1 wk resulted in marked stimulation of hair growth in the SKH-1 hairless mice (Fig. 1
). Relative to controls, mice treated with PTH (7–34) had 216% longer hairs (P < 0.001, Table 1 and Fig. 2), 40% more visible hairs (P < 0.001), and 43% more hair follicles stained with BrdU (P < 0.01). Previously we determined that mice treated with Novasome vehicle alone did not differ from untreated mice with regard to hair growth (11).
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FIG. 1. SKH-1 hairless mice treated for 1 wk. A, A control mouse after 1 wk of topical treatment with vehicle alone. B, A mouse after 1 wk of treatment with 10 µg daily topical PTH (7–34).
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FIG. 2. Topical PTH (7–34) markedly stimulated hair growth on SKH-1 hairless mice. A, Hair length was increased 216%. B, Hair number was increased 40%. C, The number of BrdU-stained hair follicles increased 43%. CTL, Animals treated with vehicle alone (n = 5); PTH (7–34), animals treated topically with 10 µg PTH (7–34) daily (n = 5). Data are presented as mean percent (relative to control) ± SEM. ***, P < 0.001; * P < 0.05.
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Serum calcium was measured in both PTH (7–34)-treated mice and control mice. There was no statistically significant difference in serum calcium concentration between the two groups.
Topical PTH (7–34) stimulated epidermal proliferation
Daily topical application of 10 µg PTH (7–34) for 1 wk resulted in marked stimulation of epidermal proliferation (Table 1
and Fig. 3). Surrogates of epidermal proliferation were significantly greater in mice receiving topical PTH (7–34) relative to control mice. In treated mice, 3H-thymidine incorporation into the epidermis was 37% greater (P < 0.05), epidermal keratinocyte nuclei staining for BrdU was 107% greater (P < 0.01, Fig. 4), and epidermal thickness was 25% greater (P < 0.005). Previously we determined that mice treated with Novasome alone did not differ from untreated mice with regard to epidermal proliferation (11). In a confirmatory experiment with C57BL/6 mice, the same topical PTH (7–34) treatment increased 3H-thymidine incorporation into epidermis 46% relative to control animals.
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FIG. 3. Topical PTH (7–34) markedly stimulated epidermal proliferation. A, 3H-thymidine incorporation into epidermis increased 37%. B, The number of BrdU-stained epidermal nuclei was increased 107%. C, Epidermal thickness was increased 25%. CTL, Animals treated with vehicle alone (n = 5); PTH (7–34), animals treated topically with 10 µg PTH (7–34) daily (n = 5). Data are presented as mean percent (relative to control) ± SEM. ** P < 0.01; * P < 0.05.
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FIG. 4. Histological preparations showing BrdU staining. A, A representative BrdU-stained section of epidermis from a control mouse. B, A representative BrdU-stained section of epidermis from a PTH (7–34)-treated (Rx’d) mouse. Red nuclei are BrdU stained (open arrow). Blue nuclei are not BrdU stained (closed arrow).
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Discussion
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One of the biggest hurdles in developing peptides for the treatment of hair growth disorders is that there has been no effective method of delivering them topically. In the current study, we formulated PTH (7–34) in a novel liposome cream, which dramatically stimulated hair growth. This represents the first demonstration that a topical peptide preparation can effectively stimulate hair growth.
PTHrP has been implicated as a possible chalone, the hypothesized but elusive hair growth inhibitor that gradually accumulates during anagen and moves the follicle to catagen when present in sufficient concentrations (6, 12) A putative mechanism for PTH (7–34) action is competitive inhibition of the PTHrP antiproliferative and prodifferentiating effects (8).
A unique aspect of skin, is the possibility to directly target it via topical treatment. Our group mixed the PTH/PTHrP receptor agonist, PTH (1–34), into Novasome A and applied the compound topically to humans (13). The antiproliferative properties of PTH (1–34) resulted in successful treatment of the hyperproliferative skin disorder, psoriasis. Whether increased epidermal proliferation caused by PTH (7–34) could exacerbate hyperproliferative skin disease, like psoriasis, remains a topic for future study.
Although the reported study was done with female mice, similar experiments have been done with male mice. The results were the same, consistent with the cutaneous effects of PTH (7–34) being similar in both sexes.
SKH-1 hairless mice were chosen for the study because they provide a background on which changes in hair growth are well discerned. SKH-1 mice lose all external hair in their first hair cycle after birth. Their complete hair loss is related to a mutation in the hairless gene (hr). Although SKH-1 mice appear to have complete hair loss, close inspection reveals that a small number of their hair follicle appear normal histologically.
The SKH-1 mouse model was chosen in part because they are amenable to close visual inspection without shaving or depilating. The avoidance of hair removal procedures removed a potential source of skin injury from our study. In addition, it was straightforward to inspect the mice and confirm the absence of inflammation or injury. In that way, we could attribute the results to anagen and not inflammation.
Endocrinologists see both men and women with alopecia that may be associated with gonadal hormone deficiencies. There are few options available for treatment. Our current study is the first to report the hair stimulating effect of a PTH/PTHrP receptor antagonist topically applied to skin in vivo. Thus, we introduce a novel paradigm to develop topical PTH analogs for treating disorders of hair growth.
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Footnotes
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This work was supported in part by A&D Bioscience, Inc. (Sudbury, MA), IGI, Inc. (Buena, NJ), and Small Business Technology Transfer Program Grant R42CA71119 (to M.F.H.).
Disclosure Statement: The authors have nothing to disclose.
First Published Online December 14, 2006
Abbreviation: BrdU, 5-Bromo-2'-deoxyuridine.
Received October 27, 2006.
Accepted for publication December 7, 2006.
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References
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Abstract
Introduction
